Compositions for producing glucose syrups

ABSTRACT

The present invention relates to a method of making glucose syrup from liquefied starch comprising, (a) contacting the liquefied starch with a glucoamylase, a pullulanase, and optionally an alpha-amylase wherein the ratio of pullulanase dose expressed as NPUN/gDS, to alpha-amylase dose expressed as FAU(A)/gDS is at least 60, particularly at least 75, particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 250, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 800 or if no alpha-amylase is present the pullulanse is present in a dose of at least 0.5, particularly at least 0.75, particularly at least 1.0, particularly at least 1.5 NPUN/gDS, and (b) saccharifying the liquefied starch.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/116,006filed on Aug. 2, 2016, now U.S. Pat. No. 10,337,041, which is a 35U.S.C. 371 national application of international application no.PCT/EP2015/052545 filed Feb. 6, 2015, which claims priority or thebenefit under 35 U.S.C. 119 of European application nos. 14195687.0 and14154239.9 filed Dec. 1, 2014 and Feb. 7, 2014, respectively. Thecontent of each application is fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions comprising analpha-amylase, a pullulanase and a glucoamylase. Furthermore, thepresent invention relates to methods of producing glucose syrupcomprising high % DX.

Description of the Related Art

Starch usually consists of about 80% amylopectin and 20% amylose.Amylopectin is a branched polysaccharide in which linear chainsalpha-1,4 D-glucose residues are joined by alpha-1,6-glucosidiclinkages. Amylopectin is partially degraded by alpha-amylase, whichhydrolyzes the 1,4-alpha-glucosidic linkages to produce branched andlinear oligosaccharides.

Alpha-amylases are used commercially for a variety of purposes such asin the initial stages of starch processing (e.g., liquefaction).Prolonged degradation of amylopectin by alpha-amylase results in theformation of so-called alpha-limit dextrins that are not susceptible tofurther hydrolysis by the alpha-amylase. Alpha-amylases (1,4-α-D-glucanglucanohydrolase, EC 3.2.1.1) constitute a group of enzymes whichcatalyze hydrolysis of starch and other linear and branched1,4-glucosidic oligo- and polysaccharides.

Branched oligosaccharides can be hydrolyzed into linear oligosaccharidesby a debranching enzyme. The remaining branched oligosaccharides can bedepolymerized to D-glucose by glucoamylase, which hydrolyzes linearoligosaccharides into D-glucose.

Debranching enzymes which can attack amylopectin are divided into twoclasses: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41),respectively. Isoamylase hydrolyzes alpha-1,6-D-glucosidic branchlinkages in amylopectin and beta-limit dextrins and can be distinguishedfrom pullulanases by the inability of isoamylase to attack pullulan, andby their limited action on alpha-limit dextrins.

It is well-known in the art to add isoamylases or pullulanases in starchconversion processes. Pullulanase is a starch debranching enzyme havingpullulan 6-glucano-hydrolase activity (EC3.2.1.41) that catalyzes thehydrolysis the α-1,6-glycosidic bonds in pullulan, releasing maltotriosewith reducing carbohydrate ends. Usually pullulanase is used incombination with an alpha amylase and/or a glucoamylase.

Pullulanases are known in the art. U.S. Pat. Nos. 6,074,854 and5,817,498 disclose a pullulanase from Bacillus deramificans. WO2009/075682 disclose a pullulanase derived from Bacillusacidopullulyticus.

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is anenzyme, which catalyzes the release of D-glucose from the non-reducingends of starch or related oligo- and polysaccharide molecules.Glucoamylases are produced by several filamentous fungi and yeast, withthose from Aspergillus, Talaromyces, Penicillium, and Trametes beingparticularly commercially important.

Commercially, glucoamylases are used to convert starchy material, whichis already partially hydrolyzed by an alpha-amylase and, e.g., apullulanase, to glucose in the form of syrup.

Before the enzymatic treatment the starch material, such as wholegrains, may be reduced in particle size, e.g., by milling, in order toopen up the structure and allowing for further processing. In drymilling whole kernels are milled and used. Wet milling gives a goodseparation of germ and meal (starch granules and protein) and is oftenapplied at locations where the starch hydrolyzate is used in theproduction of, e.g., syrups. Both dry and wet milling is well known inthe art of starch processing and may be used in a process of theinvention.

After milling, typically the starch material is liquefied. Liquefactionis carried out in the presence of an alpha-amylase, preferably, abacterial alpha-amylase and/or acid fungal alpha-amylase.

During liquefaction, the long-chained starch is degraded into branchedand linear shorter units (maltodextrins) by an alpha-amylase.Liquefaction may be carried out as a three-step hot slurry process. Theliquefaction process is carried out at between 70-95° C., such as 80-90°C., such as around 85° C., for about 10 minutes to 5 hours, typicallyfor 1-2 hours. After such treatment, the liquefied starch will typicallyhave a “dextrose equivalent” (DE) of 10-15.

Generally, liquefaction and liquefaction conditions are well known inthe art.

For the production of glucose syrup, the liquefied starch material issaccharified. In a typical saccharification process, maltodextrinsproduced during liquefaction are converted into dextrose by adding aglucoamylase and a debranching enzyme, such as an isoamylase (U.S. Pat.No. 4,335,208) or a pullulanase. The temperature is lowered to 60° C.,prior to the addition of the glucoamylase and debranching enzyme. Thesaccharification process proceeds for 24-72 hours. Prior to addition ofthe saccharifying enzymes, the pH is reduced to below 4.5, whilemaintaining a high temperature (above 95° C.), to inactivate theliquefying alpha-amylase.

For the production of syrup, enzyme compositions used should at leastcomprise a glucoamylase and a pullulanase, however, often alpha-amylaseactivity will also be present, e.g., when using Aspergillus nigerglucoamylase the A. niger alpha-amylase from the production host willalso be present in the composition. It has surprisingly been found thatin order to reach high % DX values of the syrup, e.g., above 95%, thelevel of alpha-amylase present in the composition should be carefullycontrolled.

The present invention provides compositions and methods for producinghigh glucose syrups having a % DX of around 96%.

Applications for higher DX syrups are: production of DMH (dextrosemonohydrate), fermentation chemicals such as organic acids (such ascitric acid, lactic acid, etc.) or amino acids (such as L-lysine,L-threonine, L-tryptophane, monosodium glutamate and L-cysteine), Highfructose corn syrups, crystalline fructose and other specialty syrups.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a compositioncomprising an alpha-amylase, a pullulanase and a glucoamylase, whereinthe ratio of pullulanase dose expressed as NPUN/g, to alpha-amylase doseexpressed as FAU(A)/g or a KNU/g is at least 60.

In a second aspect, the present invention relates to a method of makingglucose syrup from liquefied starch comprising, (a) contacting theliquefied starch with a glucoamylase, a pullulanase, and optionally analpha-amylase, wherein the ratio of pullulanase dose expressed asNPUN/gDS, to alpha-amylase dose expressed as FAU(A)/gDS or as KNU/gDS isat least 60, particularly at least 75, particularly at least 100, moreparticularly at least 150, more particularly at least 200, moreparticularly at least 250, more particularly at least 300, moreparticularly at least 400, more particularly at least 500, moreparticularly at least 600, more particularly at least 800 or if noalpha-amylase present the pullulanase is present in a dose of at least0.5, particularly at least 0.75, particularly at least 1.0, particularlyat least 1.5 NPUN/gDS, and (b) saccharifying the liquefied starch.

Definitions

Alpha-amylase: Alpha-amylases (1,4-alpha-D-glucan glucanohydrolase, E.C.3.2.1.1) are a group of enzymes which catalyze the hydrolysis of starchand other linear and branched 1,4 glucosidic oligo- and polysaccharides.Alpha-amylases used according to the present invention may be obtainedfrom fungal or bacterial sources. For purposes of the present invention,fungal alpha amylase activity can be determined as FAU(A) using thealpha amylase assay described in the Materials and Methods. Activity ofbacterial alpha-amylases can be determined as Kilo Novo alpha-amylaseUnits (KNU) according to the procedure described in the paragraph “KiloNovo alpha-amylase Units (KNU)” below.

Acid alpha-Amylase Units (FAU(A)): Acid alpha-amylase activity may bemeasured in FAU(A) (Acid Fungal Alpha-amylase Units). 1 FAU(A) isdefined as the amount of enzyme which degrades 5.260 mg starch drymatter per hour under the standard conditions specified in the table“First reaction, starch degradation” below.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

FAU(A), the acid alpha-amylase activity is determined in accordance withthe following description. The principle of the reaction is based on thetwo steps. In the first step, the enzyme acid alpha-amylase hydrolyzesstarch into different oligosaccharides. In the second step, iodine formsa blue complex with starch but not with its degradation products. Theintensity of color is therefore directly proportional to theconcentration of starch. The activity is determined using reversecolorimetry as a reduction in the concentration of starch underspecified analytic conditions.

First reaction, starch degradation Substrate Starch, approx. 0.3 g/LBuffer Citrate, approx. 0.05M CaCl2, 1.85 mM pH 2.50 ± 0.05 Incubationtemperature 37° C. Reaction time 180 seconds Enzyme working range0.01-0.04 FAU(A)/mL Second reaction, starch-iodine complex Iodine 0.0432g/L Incubation temperature 37° C. Reaction time 60 seconds Wavelength600 nmKilo Novo Alpha-Amylase Units (KNU)

Bacterial alpha-amylase activity may be determined using potato starchas substrate. The method is based on breakdown of starch in solution byamylase and the fact that starch gives a blue-black color in thepresence of iodine. As the enzyme reaction proceeds, aliquots of thereaction are withdrawn and analyzed for their starch content by mixingwith an iodine solution. As starch is broken down, the blue-black colorin the presence of iodine fades and gradually turns into a reddish-browncolor. This is compared with a colored glass standard. The end point isreached when the color matches the glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions as defined in the “KNU” tablebelow (i.e., at 37° C.+/−0.05; 0.0003 M Ca²⁺; and pH 5.6) dextrinizes5260 mg/hour starch dry substance; e.g., Merck Amylum solubile.

KNU Temperature 37 ± 0.05° C. pH 5.6 Substrate concentration 4.63 mg dryweight/mL Reaction time 7-20 minutes, up to 1 hour Ca²⁺ concentrationapprox. 0.0003M for reaction mix containing 2 mL sample solution

Pullulanase: The term “pullulanase” means a starch debranching enzymehaving pullulan 6-glucano-hydrolase activity (EC3.2.1.41) that catalyzesthe hydrolyses the α-1,6-glycosidic bonds in pullulan, releasingmaltotriose with reducing carbohydrate ends. For purposes of the presentinvention, pullulanase activity is determined as NPUN according to theprocedure described in the Materials and Methods and in the followingparagraph.

Pullulanase activity (NPUN): The NPUN (New Pullulanase Unit Novozymes)is a unit of endopullulanase activity measured in the followingprocedure.

1 NPUN=One pullulanase unit (NPUN) is defined as the enzyme amount,which releases reducing ends equivalent to 0.35 μmol glucose per minuteunder the standard conditions specified in the table “First reaction,pullulan degradation” below.

In the first reaction, the substrate is equally present in both samplemain and sample blank. However, the reaction of sample main is performedat pH 5.0, while there is no reaction in the sample blank at pH 9.6,where neither pullulanases nor amyloglucosidases (glucoamylase) areenzymatically active.

First reaction, pullulan degradation Substrate BH4 reduced pullulan, 5.3g/L Buffer (main) Acetate, approx. 0.1M EDTA, 5.3 mM Acarbose, 0.018%(if sample contains glucoamylase) pH (main) 5.0 Buffer (blank) CHES, 42mM acetate, 17 mM EDTA, 5.3 mM pH (blank) 9.6 Incubation temperature 50°C. Reaction time 540 seconds Enzyme working range 0.03-0.15 NPUN/mL

In the second reaction, the pH is adjusted to approx. 9.6 and theglucose in samples is phosphorylated to non-reducingD-glucose-6-phosphate by glucokinase, which has optimal activity andstability in this range and is specific to glucose at pH 9 (ref. Goward,1986, Biochem. J. 237: 415-420). This step depends on identical pH insample main and sample blank to remove equal amounts of glucose in both.

Second reaction, background glucose elimination Substrate glucose insample, after first reaction Buffer CHES, 58 mM (main) or 76 mM (blank)acetate, 43 mM (main) or 7.2 mM (blank) EDTA, 2.2 mM ATP, 1.11 mg/mlMgCl₂, 4.4 mM Glucokinase 0.11 U/ml pH approx. 9.6 Incubationtemperature 50° C. Reaction time 720 seconds

Glucoamylase: The term glucoamylase (1,4-alpha-D-glucan glucohydrolase,EC 3.2.1.3) is defined as an enzyme, which catalyzes the release ofD-glucose from the non-reducing ends of starch or related oligo- andpolysaccharide molecules. For purposes of the present invention,glucoamylase activity is determined as AGU according to the proceduredescribed in the Materials and Methods and in the following paragraph.

Glucoamylase activity (AGU): The Glucoamylase Unit (AGU) is defined asthe amount of enzyme, which hydrolyzes 1 micromole maltose per minute ina 0.1 M acetate buffer at an incubation temperature 37° C., a pH of 4.3,a maltose starting concentration of 100 mM, and a reaction time of 6minutes, thereby generating alpha-D-glucose. The definition applies toan enzyme working range of 0.5-4.0 AGU/m L.

After incubation, the reaction may be stopped with NaOH and the amountsof glucose measured using the following two-step color reaction method:Glucose is phosphorylated by ATP, in a reaction catalyzed by hexokinase.The glucose-6-phosphate formed is oxidized to 6-phosphogluconate byglucose-6-phosphate dehydrogenase. In this same reaction, an equimolaramount of NAD+ is reduced to NADH with a resulting increase inabsorbance at 340 nm. Reaction conditions are as specified in the tablebelow:

Color reaction Tris approx. 35 mM ATP 0.7 mM NAD⁺ 0.7 mM Mg²⁺ 1.8 mMHexokinase >850 U/L Glucose-6-P-DH >850 U/L pH approx. 7.8 Temperature37.0° C. ± 1.0° C. Reaction time 420 seconds Wavelength 340 nm

Degree of polymerization (DP): DP refers to the number (n) ofanhydroglucopyranose units in a given saccharide. Examples of DP1 aremonosaccharides, such as glucose and fructose. DP2 are disaccharides,such as maltose and sucrose.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. According to some embodiments, themature polypeptide of SEQ ID NO: 6 consists essentially of amino acids18 to 573 of SEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 7consists essentially of amino acids 18 to 573 of SEQ ID NO: 7, themature polypeptide of SEQ ID NO: 8 consists essentially of amino acids18 to 573 of SEQ ID NO: 8, the mature polypeptide of SEQ ID NO: 5consists essentially of amino acids 18 to 573 of SEQ ID NO: 9, themature polypeptide of SEQ ID NO: 10 consists essentially of amino acids18 to 573 of SEQ ID NO: 10, the mature polypeptide of SEQ ID NO: 11consists essentially of amino acids 18 to 573 of SEQ ID NO: 11, themature polypeptide of SEQ ID NO: 12 consists essentially of amino acids18 to 573 of SEQ ID NO: 12, the mature polypeptide of SEQ ID NO: 13consists essentially of amino acids 18 to 576 of SEQ ID NO: 13, themature polypeptide of SEQ ID NO: 14 consists essentially of amino acids18 to 576 of SEQ ID NO: 14.

In particular, the mature polypeptide of SEQ ID NO: 6 may consist ofamino acids 18 to 573 of SEQ ID NO: 6.

The mature polypeptide of SEQ ID NO: 7 may consist of amino acids 18 to573 of SEQ ID NO: 7.

The mature polypeptide of SEQ ID NO: 8 may consist of amino acids 18 to573 of SEQ ID NO: 8.

The mature polypeptide of SEQ ID NO: 9 may consist of amino acids 18 to573 of SEQ ID NO: 9.

The mature polypeptide of SEQ ID NO: 10 may consist of amino acids 18 to573 of SEQ ID NO: 10.

The mature polypeptide of SEQ ID NO: 11 may consist of amino acids 18 to573 of SEQ ID NO: 11.

The mature polypeptide of SEQ ID NO: 12 may consist of amino acids 18 to573 of SEQ ID NO: 12.

The mature polypeptide of SEQ ID NO: 13 may consist of amino acids 18 to576 of SEQ ID NO: 13.

The mature polypeptide of SEQ ID NO: 14 may consist of amino acids 18 to576 of SEQ ID NO: 14.

In further embodiments, the mature polypeptide of SEQ ID NO: 6 consistsof amino acids 18 to 573 of SEQ ID NO: 6, the mature polypeptide of SEQID NO: 7 consists of amino acids 18 to 573 of SEQ ID NO: 7, the maturepolypeptide of SEQ ID NO: 8 consists of amino acids 18 to 573 of SEQ IDNO: 8, the mature polypeptide of SEQ ID NO: 9 consists of amino acids 18to 573 of SEQ ID NO: 9, the mature polypeptide of SEQ ID NO: 10 consistsof amino acids 18 to 573 of SEQ ID NO: 10, the mature polypeptide of SEQID NO: 11 consists of amino acids 18 to 573 of SEQ ID NO: 11, the maturepolypeptide of SEQ ID NO: 12 consists of amino acids 18 to 573 of SEQ IDNO: 12, the mature polypeptide of SEQ ID NO: 13 consists of amino acids18 to 576 of SEQ ID NO: 13, and the mature polypeptide of SEQ ID NO: 14consists of amino acids 18 to 576 of SEQ ID NO: 14.

The prediction of mature polypeptide sequences may be based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 17 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13 and SEQ ID NO: 14 are a signal peptide. The sequence defined by aminoacids 19 to 474 (particularly 19 to 471) of SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:12 or amino acids 19 to 471 of SEQ ID NO: 13 or of SEQ ID NO: 14 is thecatalytic domain. The sequence defined by amino acids 480 to 573 of SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11 or SEQ ID NO: 12 or amino acids 483 to 576 of SEQ ID NO: 13 orSEQ ID NO: 14 is a starch binding domain.

According to other embodiments, the mature polypeptide of SEQ ID NO: 18is defined by amino acids 22 to 450 of SEQ ID NO: 18.

In further embodiments, the mature peptide of SEQ ID NO: 19 is definedby amino acids 22-471 of SEQ ID NO: 19, whereas amino acids 1-21 are asignal peptide.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the −nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for producinghigh glucose syrup, in particular, glucose syrups having % DX (% DP1)values above 95%.

It has surprisingly been found that in order to reach high % DX valuesof the syrup, e.g., above 95%, the level of alpha-amylase present in thecomposition should be carefully controlled.

The methods and compositions according to the invention provides glucosesyrups with higher % DX when alpha-amylase levels are optimized so thatat a given ratio of NPUN to AGU the NPUN/FAU(A) or NPUN/KNU is adjustedto be at least 60, particularly at least 80. The high % DX syrups can beobtained at industrially relevant substrate concentrations—typicallymore than 30% initial dry solids content (DS).

In one embodiment, the present invention relates to a method of makingglucose syrup from liquefied starch comprising, (a) contacting theliquefied starch with a glucoamylase, a pullulanase, and optionally analpha-amylase wherein the ratio of pullulanase dose expressed asNPUN/gDS, to alpha-amylase dose expressed as FAU(A)/gDS or KNU/gDS is atleast 60, particularly at least 75, particularly at least 100, moreparticularly at least 150, more particularly at least 200, moreparticularly at least 250, more particularly at least 300, moreparticularly at least 400, more particularly at least 500, moreparticularly at least 600, more particularly at least 800 or if noalpha-amylase present the pullulanase is present in a dose of at least0.5, particularly at least 0.75, particularly at least 1.0, particularlyat least 1.5 NPUN/gDS, and (b) saccharifying the liquefied starch.

In some embodiments, the method comprises contacting the liquefiedstarch with a glucoamylase, a pullulanase, and optionally analpha-amylase wherein the ratio of pullulanase dose expressed asNPUN/gDS, to alpha-amylase dose expressed as FAU(A)/gDS or KNU/gDS iswithin the range of 100-700, such as within the range of 200-600, suchas within the range of 300-500, such as within the range of 350-500,such as within the range of 375-475, or such as within the range of400-450.

According to other embodiments, the method comprises contacting theliquefied starch with a glucoamylase, a pullulanase, and optionally analpha-amylase, wherein the dose of alpha amylase is within the range of0-0.008 FAU(A)/gDS and the dose of pullulanase is within the range of0.5-1.5 NPUN/gDS.

In other embodiments, the dose of alpha amylase is within the range of0-0.007 FAU(A)/g DS and the dose of pullulanase is within the range of0.6-1.4 NPUN/g DS. In other embodiments the dose of alpha amylase iswithin the range of 0-0.006 FAU(A)/gDS and the dose of pullulanase iswithin the range of 0.7-1.3 NPUN/gDS.

In other embodiments, the dose of alpha amylase is within the range of0-0.005 FAU(A)/gDS and the dose of pullulanase is within the range of0.75-1.25 NPUN/gDS.

In other embodiments, the dose of alpha amylase is within the range of0-0.004 FAU(A)/gDS and the dose of pullulanase is within the range of0.8-1.2 NPUN/gDS.

In other embodiments, the dose of alpha amylase is within the range of0-0.003 FAU(A)/gDS and the dose of pullulanase is within the range of0.9-1.1 NPUN/gDS.

In other embodiments, the dose of alpha amylase is within the range of0-0.0025 FAU(A)/g DS and the dose of pullulanase is within the range of0.95-1 NPUN/g DS.

The ratio between pullulanase expressed as NPUN/gDS and glucoamylaseexpressed as AGU/gDS may in particular be within the range of 2-15, suchas within the range of 2-10, within the range of 2-5, within the rangeof 3-5 or within the range of 3.5-4.

According to other embodiments, the method comprises contacting theliquefied starch with a glucoamylase, a pullulanase, and optionally analpha-amylase, wherein the dose of pullulanase is within the range of0.5-1.5 NPUN/gDS and the dose of glucoamylase is within the range of0.125-0.375 AGU/gDS.

In other embodiments, the dose of pullulanase is within the range of0.6-1.4 NPUN/gDS and the dose of glucoamylase is within the range of0.15-0.35 AGU/gDS.

In other embodiments, the dose of pullulanase is within the range of0.7-1.3 NPUN/gDS and the dose of glucoamylase is within the range of0.175-0.325 AGU/gDS.

In other embodiments, the dose of pullulanase is within the range of0.8-1.2 NPUN/gDS and the dose of glucoamylase is within the range of0.2-0.3 AGU/gDS.

At low levels of glucoamylase longer saccharification times may beneeded. In one embodiment, the glucoamylase dose, expressed as AGU/gDS,is at least 0.1, particularly at least 0.15, particularly at least 0.18,particularly at least 0.2, more particularly at least 0.22, moreparticularly at least 0.23, more particularly at least 0.25, even moreparticularly at least 0.28.

Using the method and compositions according to the invention very high %DX values can be obtained. In a particular embodiment, the glucose syrupcomprises a DP1 (% DX) of at least 95.8%, particularly at least 95.9%,particularly at least 96%, more particularly at least 96.1%.

Saccharification times may vary depending on enzyme dose. In oneparticular embodiment, the saccharification time is at least 24 hours,at least 30 hours, at least 36 hours, at least 48 hours, at least 54hours, at least 60 hours, at least 72 hours.

In the process according to the invention, the starchhydrolysis/saccharification may in particular take place at a pH whichis within the range of 3.5-5.0, such as at pH in the range of 4.0-4.5,and at a temperature, which is within the range of 59-70° C., such as inthe range of 59-65° C. or such as in the range of 59-62° C.

The liquefied starch used as substrate for the saccharification processaccording to the invention may be a starch slurry or partiallyhydrolyzed starch (liquefact or maltodextrin). In particular, the instarch slurry or partly hydrolyzed starch may have a Dextrose equivalent(DE) in the range of 5-42, such as in the range of 5-30, in the range of8-18 or such as in the range of 9-14.

The starch may be from any source, in particular, corn, wheat ortapioca. The starch slurry or partially hydrolysed starch may haveresidual alpha amylase activity from the liquefaction process present orit may have been deactivated, such as by reducing the pH to below 4.5,while maintaining a high temperature (above 95° C.), to inactivate theliquefying alpha-amylase.

The conductivity of said starch slurry or partially hydrolysed starchmay in particular be within the range of 0-500 microS/cm. According tosome embodiments, the calcium content corresponds to 0-200 ppm freecalcium.

In the process according to the invention, the starchhydrolysis/saccharification may in particular take place at a pH whichis within the range of 3.5-5.0, such as at pH in the range of 4.0-4.7,and at a temperature, which is within the range of 58-70° C., such as inthe range of 58-65° C., in the range of 59-65° C. or such as in therange of 59-62° C.

The composition comprising liquefied starch provided as a startingmaterial; i.e., composition comprising liquefied starch provided the instep i) of the process may contain from 25-45% dry solids (% DS), suchas from 25-40% DS.

The method according to the invention is applicable at industry relevantsubstrate doses. In one embodiment the initial dry solids content (DS)in the liquefied starch substrate is at least 25%, particularly at least30%, more particularly at least 35%, even more particularly at least40%.

Enzyme Compositions

The present invention also relates to compositions comprising aglucoamylase, a pullulanase and an alpha-amylase.

In a particular embodiment, the composition comprises an alpha-amylase,a pullulanase and a glucoamylase, wherein the ratio of pullulanase doseexpressed as NPUN/g, to alpha-amylase dose expressed as FAU(A)/g or asKNU/g is at least 60.

More particularly, the composition comprises an alpha-amylase, apullulanase and a glucoamylase, wherein the ratio of pullulanase doseexpressed as NPUN/g, to alpha-amylase dose expressed as FAU(A)/g or asKNU/g is at least 75, particularly at least 100, more particularly atleast 150, more particularly at least 200, more particularly at least250, more particularly at least 300, more particularly at least 400,more particularly at least 500, more particularly at least 600, moreparticularly at least 800.

In another embodiment, the ratio of pullulanase dose expressed asNPUN/g, to alpha-amylase dose expressed as FAU(A)/g or as KNU/g is inthe range from 60-1000, more particularly 70-800, more particularly80-600, more particularly 90-500, more particularly 100-400.

The ratio of pullulanase dose expressed as NPUN/g, to alpha-amylase doseexpressed as FAU(A)/g or KNU/g may in particular be within the range of100-700, such as within the range of 200-600, such as within the rangeof 300-500, such as within the range of 350-500, such as within therange of 375-475, or such as within the range of 400-450.

In a further aspect, the invention relates to a composition, wherein theratio between pullulanase expressed as NPUN/g and glucoamylase expressedas AGU/g is at least 2, particularly at least 2.5, particularly at least3, more particularly at least 3.5, more particularly at least 5, moreparticularly at least 10, even more particularly at least 15.

The ratio between pullulanase expressed as NPUN/g and glucoamylaseexpressed as AGU/g may in particular be within the range of 2-15, suchas within the range of 2-10, within the range of 2-5, within the rangeof 3-5 or within the range of 3.5-4.

Pullulanase

Any pullulanase may be used in a process of the present invention. In anembodiment, the pullulanase is a pullulanase from Bacillus deramificans,e.g., disclosed in U.S. Pat. Nos. 6,074,854 and 5,817,498, or apullulanase derived from Bacillus acidopullulyticus, e.g., disclosed inWO 2009/075682 (SEQ ID NO: 4; GENESEQP: AXB71624).

Commercially available pullulanases include Promozyme D2 available fromNovozymes A/S, Bagsvaerd, Denmark), Novozym 26062 (Novozymes) andOptimax L 1000 (DuPont-Genencor)

Glucoamylase

A glucoamylase used according to the invention may be derived from anysuitable source, e.g., derived from a microorganism or a plant.Preferred glucoamylases are of fungal or bacterial origin, selected fromthe group consisting of Aspergillus glucoamylases, in particular A.niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5):1097-1102), or variants thereof, such as those disclosed in WO 92/00381,WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamoriglucoamylase disclosed in WO 84/02921, A. oryzae glucoamylase (Agric.Biol. Chem., 1991, 55(4): 941-949), or variants or fragments thereof.Other Aspergillus glucoamylase variants include variants with enhancedthermal stability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9:499-505); D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582);N182 (Chen et al., 1994, Biochem. J. 301: 275-281); disulphide bonds,A246C (Fierobe et al., 1996, Biochemistry 35: 8698-8704; andintroduction of Pro residues in position A435 and S436 (Li et al., 1997,Protein Eng. 10: 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 andNagasaka et al., 1998, “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, ApplMicrobiol Biotechnol 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, andTalaromyces thermophilus (U.S. Pat. No. 4,587,215).

Contemplated fungal glucoamylases include Trametes cingulata, disclosedin WO 2006/069289.

In an embodiment, the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus as described in WO2011/066576 (SEQ ID NOs 2, 4 or 6), or from a strain of the genusGloephyllum, in particular a strain of Gloephyllum as described in WO2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16) or a strain of thegenus Nigrofomes, in particular a strain of Nigrofomes sp. disclosed inWO 2012/064351 (SEQ ID NO: 2) (all references hereby incorporated byreference) or a strain of Penicillium, in particular Penicilliumoxalicum disclosed in WO 2011/127802 (SEQ ID NO: 2) or WO 2013/036526.Contemplated are also glucoamylases which exhibit a high identity to anyof the above-mentioned glucoamylases, i.e., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, such as 100% identity to any oneof the mature parts of the enzyme sequences mentioned above.

In an embodiment, the glucoamylase is derived from a strain of the genusTrichoderma, in particular, as described in WO 2009/048487, WO2009/048488, WO 2008/045489, WO 2011/022465, WO 2012/001139.

Commercially available glucoamylase compositions include AMG 200L; AMG300L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL,SPIRIZYME™ B4U, SPIRIZYME ULTRA™, SPIRIZYME EXCEL™ and AMG™ E (fromNovozymes A/S, Denmark); OPTIDEX™ 300, GC480™ and GC147™ (from GenencorInt., USA); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™and G990 ZR (from DuPont-Genencor).

Alpha-Amylase

Fungal alpha-amylases include alpha-amylases derived from a strain ofthe genus Aspergillus, such as, Aspergillus oryzae, Aspergillus nigerand Aspergillus kawachii alpha-amylases.

A preferred acidic fungal alpha-amylase is a Fungamyl-like alpha-amylasewhich is derived from a strain of Aspergillus oryzae. According to thepresent invention, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e., more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acidic alpha-amylase is derived from a strainAspergillus niger. In a preferred embodiment, the acid fungalalpha-amylase is the one from A. niger disclosed as “AMYA_ASPNG” in theSwiss-prot/TeEMBL database under the primary accession no. P56271 anddescribed in WO 89/01969 (Example 3).

Other contemplated wild-type alpha-amylases include those derived from astrain of the genera Rhizomucor and Meripilus, preferably a strain ofRhizomucor pusillus (WO 2004/055178 incorporated by reference) orMeripilus giganteus.

In a preferred embodiment, the alpha-amylase is derived from Aspergilluskawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81:292-298, “Molecular-cloning and determination of the nucleotide-sequenceof a gene encoding an acid-stable alpha-amylase from Aspergilluskawachii”; and further as EMBL:#AB008370.

The fungal alpha-amylase may also be a wild-type enzyme comprising astarch-binding domain (SBD) and an alpha-amylase catalytic domain (i.e.,non-hybrid), or a variant thereof. In an embodiment the wild-typealpha-amylase is derived from a strain of Aspergillus kawachii.

Fungal Hybrid Alpha-Amylase

In a preferred embodiment, the alpha amylase is a fungal acidalpha-amylase is a hybrid alpha-amylase. Preferred examples of fungalhybrid alpha-amylases include the ones disclosed in WO 2005/003311 orU.S. application publication no. 2005/0054071 (Novozymes) or U.S.application No. 60/638,614 (Novozymes) which is hereby incorporated byreference. A hybrid alpha-amylase may comprise an alpha-amylasecatalytic domain (CD) and a carbohydrate-binding domain/module (CBM),such as a starch binding domain, and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include thosedisclosed in Tables 1 to 5 of the examples in U.S. application No.60/638,614, including Fungamyl variant with catalytic domain JA118 andAthelia rolfsii SBD (SEQ ID NO:100 in U.S. 60/638,614), Rhizomucorpusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ IDNO:101 in U.S. 60/638,614), Rhizomucor pusillus alpha-amylase withAspergillus niger glucoamylase linker and SBD (which is disclosed inTable 5 as a combination of amino acid sequences SEQ ID NO: 20, SEQ IDNO: 72 and SEQ ID NO: 96 in U.S. application Ser. No. 11/316,535) or asV039 in Table 5 in WO 2006/069290, and Meripilus giganteus alpha-amylasewith Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in U.S.application No. 60/638,614). Other specifically contemplated hybridalpha-amylases are any of the ones listed in Tables 3, 4, 5, and 6 inExample 4 in U.S. application Ser. No. 11/316,535 and WO 2006/069290(hereby incorporated by reference).

Other specific examples of contemplated hybrid alpha-amylases includethose disclosed in U.S. application publication no. 2005/0054071,including those disclosed in Table 3 on page 15, such as Aspergillusniger alpha-amylase with Aspergillus kawachii linker and starch bindingdomain.

Contemplated are also alpha-amylases which exhibit a high identity toany of above mention alpha-amylases, i.e., more than 70%, more than 75%,more than 80%, more than 85% more than 90%, more than 95%, more than96%, more than 97%, more than 98%, more than 99% or even 100% identityto the mature enzyme sequences.

Bacterial Alpha-Amylase

Bacterial alpha-amylases useful in the processes according to theinvention include alpha-amylases derived from a strain of the genusBacillus, such as Bacillus licheniformis and Bacillusstearothermophilus.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE™ (DSM), BAN™, TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ X, LIQUOZYME™SC and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S) and CLARASE™ L-40,000,DEX-LO™, SPEZYME™ FRED, SPEZYME™ AA, SPEZYME™ ALPHA, SPEZYME™ DELTA AA,GC358, GC980, SPEZYME™ CL and SPEZYME™ RSL (DuPont-Genencor), FUELZYME™(Verenium Corp., USA).

In a particular embodiment, the composition according to the inventioncomprises an alpha amylase, a glucoamylase (AMG), and a pullulanase, andwherein the alpha amylase is selected from Aspergillus niger orRhizomucor pusillus alpha amylases, or variants thereof, theglucoamylase is selected from Aspergillus niger, Talaromyces emersonii,Trametes cingulata or Gloeophyllum trabeum glucoamylases, or variantsthereof, and the pullulanase is selected from Bacillus deramificans orBacillus acidopullulyticus pullulanases, or hybrids and/or variantsthereof.

In further particular embodiments, the composition according to theinvention comprises a glucoamylase which comprises/consists essentiallyof/consists of an amino acid sequence selected from the group consistingof:

i) The amino acid sequence set forth in any one of SEQ ID NO: 1, 4, 6,7, 8, 9, 10, 11, 12, 13, 14 and 15 or a mature polypeptide thereof;

ii) A subsequence of the amino acid sequence set forth in any one of SEQID NOs: 1, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 or of said maturepolypeptide thereof;

iii) An amino acid sequence, which has at least 70%, such as at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, such as at least 99.5% sequenceidentity to any one of the amino acids sequences set forth in i) andii).

When the glucoamylase comprises a subsequence as defined in ii) or anamino acid sequence as defined in iii), it preferably has at least 75%,such as at least 80%, at least 85%, at least 90% or such as at least 95%of the glucoamylase activity of the respective amino acid defined in i)of which it is a subsequence or variant, when tested as set forth abovein relation to the definition of “Glucoamylase activity (AGU)”.

In other embodiments, the composition according to the inventioncomprises an alpha-amylase, which comprises/consists essentiallyof/consists of an amino acid sequence selected from the group consistingof:

i) the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 19,20, 21, 22, 23, 24 and 25 or a mature polypeptide thereof;

ii) a subsequence of the amino acid sequence set forth in any one of SEQID NOs: 2, 5, 19, 20, 21, 22, 23, 24 and 25 or of said maturepolypeptide thereof;

iii) a variant amino acid sequence, which has at least 70%, such as atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, such as at least 99.5% sequenceidentity to any one of the amino acids sequences set forth in i) andii).

When the alpha-amylase defined above is a subsequence or a variant, itpreferably has at least 75%, such as at least 80%, at least 85%, atleast 90% or such as at least 95% of the alpha-amylase activity of therespective alpha-amylase selected from SEQ ID NOs: 2, 5, 19, 20, 21, 22,23, 24 and 25 or the mature polypeptide thereof, of which it is asubsequence or variant, when tested as set forth above in relation tothe definition of “Acid Alpha-Amylase Units (FAU(A))” or “Kilo Novoalpha-amylase Units (KNU)”: In the present context an alpha-amylasecomprising an amino acid sequences selected from the group consisting ofSEQ ID NOs: 2, 5 and 19 or the mature polypeptide thereof, is consideredto be a fungal alpha amylase and activity is tested as provided inrelation to the above definition of “Acid alpha-Amylase Units (FAU(A))”.An alpha-amylase comprising an amino acid sequences selected from thegroup consisting of SEQ ID NOs: 20-25 or the mature polypeptide thereofis considered to be a bacterial alpha amylase and activity is tested asprovided in relation to the above definition of “Kilo Novo alpha-amylaseUnits (KNU).”

In particular embodiments, the alpha-amylase comprising or consisting ofthe amino acid sequence defined in iii) is a variant of an alpha-amylasecomprising or consisting of the amino sequence defined in SEQ ID NO: 20or a mature polypeptide thereof, wherein the following mutations havebeen made: I181*/G182*/N193F (using the amino acid numbering in SEQ IDNO: 20).

According to other embodiments, the alpha-amylase comprising orconsisting of the amino acid sequence defined in iii) is a variant of analpha-amylase comprising or consisting of the amino sequence defined inSEQ ID NO: 23 or a mature polypeptide thereof, wherein the followingmutations have been made: H156Y+A181T+N190F+A209V+Q264S (using the aminoacid numbering in SEQ ID NO: 21).

In even further embodiments, the alpha-amylase comprising or consistingof the amino acid sequence defined in iii) is a variant of analpha-amylase comprising or consisting of the amino sequence defined inSEQ ID NO: 23 or a mature polypeptide thereof, wherein the followingmutations have been made:G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the numberingin SEQ ID NO: 21).

In still other embodiments, the composition according to the inventioncomprises a pullulanase, which comprises/consists essentiallyof/consists of an amino acid sequence selected from the group consistingof:

i) the amino acid sequence set forth in any one of SEQ ID NOs: 3, 16, 17and 18 or a mature polypeptide thereof;

ii) a subsequence of the amino acid sequence set forth in any one of SEQID NOs: 3, 16, 17 and 18 or of said mature polypeptide thereof;

iii) an amino acid sequence, which has at least 70%, such as at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, such as at least 99.5% sequenceidentity to any one of the amino acids sequences set forth in i) andii).

When the pullulanase comprises a subsequence as defined in ii) or avariant amino acid sequence as defined in iii), it preferably has atleast 75%, such as at least 80%, at least 85%, at least 90% or such asat least 95% of the pullulanase activity of the respective amino aciddefined in i) of which it is a subsequence or variant, when tested asset forth above in relation to the definition of “Pullulanase activity(NPUN)”.

Within the scope of the present invention are embodiments wherein theglucoamylase comprises or consists of an amino acid sequence selectedfrom the group consisting of:

i) the amino acid sequence set forth in SEQ ID NO: 4 or a maturepolypeptide thereof;

ii) a subsequence of the amino acid sequence set forth in SEQ ID NO: 4or of said mature polypeptide thereof; and

iii) a variant amino acid sequence, which has at least 70%, such as atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, such as at least 99.5% sequenceidentity to any one of the amino acids sequences set forth in i) andii); wherein the pullulanase comprises or consists of an amino acidsequence selected from the group consisting of:

iv) the amino acid sequence set forth in any one of SEQ ID NOs: 3, 16,17 and 18 or a mature polypeptide thereof;

v) a subsequence of the amino acid sequence set forth in any one of SEQID NOs: 3, 16, 17 and 18 or of said mature polypeptide thereof; and

vi) a variant amino acid sequence, which has at least 70%, such as atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, such as at least 99.5% sequenceidentity to any one of the amino acids sequences set forth in iv) andv); and wherein the alpha-amylase comprises or consists of an amino acidsequence selected from the group consisting of:

vii) the amino acid sequence set forth in SEQ ID NO: 5 or a maturepolypeptide thereof;

viii) a subsequence of the amino acid sequence set forth in SEQ ID NO: 5or of said mature polypeptide thereof; and

ix) a variant amino acid sequence, which has at least 70%, such as atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, such as at least 99.5% sequenceidentity to any one of the amino acids sequences set forth in vii) andviii).

When said glucoamylase is a subsequence or a variant amino acid sequenceas defined above, it preferably has at least 75%, such as at least 80%,at least 85%, at least 90% or such as at least 95% of the glucoamylaseactivity of the respective amino acid sequence (e.g., the amino acidsequence set forth in SEQ ID NO: 4 or a mature polypeptide thereof) ofwhich it is a subsequence or variant, when tested as set forth above inrelation to the definition of “Glucoamylase activity (AGU)”.

When said pullulanase is a subsequence or a variant amino acid sequenceas defined above, it preferably has at least 75%, such as at least 80%,at least 85%, at least 90% or such as at least 95% of the pullulanaseactivity of the respective amino acid sequence (e.g., the amino acidsequence set forth in any one of SEQ ID NOs: 3, 16, 17 and 18 or amature polypeptide thereof) of which it is a subsequence or variant,when tested as set forth above in relation to the definition of“Pullulanase activity (NPUN)”.

When the alpha-amylase defined above is a subsequence or a variant, itpreferably has at least 75%, such as at least 80%, at least 85%, atleast 90% or such as at least 95% of the alpha-amylase activity of therespective alpha-amylase selected from SEQ ID NOs: 5 or of the maturepolypeptide thereof, of which it is a subsequence or variant, whentested as set forth above in relation to the definition of “Acidalpha-Amylase Units (FAU(A))”.

In a particular embodiment, the glucoamylase is selected from theglucoamylase disclosed in SEQ ID NO: 1 or the mature polypeptidethereof, and a glucoamylase having a sequence identity to thepolypeptide of SEQ ID NO: 1 or the mature polypeptide thereof, of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which hasglucoamylase activity; e.g., at least 75%, such as at least 80%, atleast 85%, at least 90% or such as at least 95% of the glucoamylaseactivity of the glucoamylase disclosed in SEQ ID NO: 1 or the maturepolypeptide thereof, when tested as set forth above in relation to thedefinition of “Glucoamylase activity (AGU)”.

In another particular embodiment, the glucoamylase is selected from theglucoamylase disclosed in SEQ ID NO: 4 or the mature polypeptidethereof, and a glucoamylase having a sequence identity to the maturepolypeptide of SEQ ID NO: 4 or the mature polypeptide thereof, of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which hasglucoamylase activity; e.g., at least 75%, such as at least 80%, atleast 85%, at least 90% or such as at least 95% of the glucoamylaseactivity of the glucoamylase disclosed in SEQ ID NO: 4 or the maturepolypeptide thereof, when tested as set forth above in relation to thedefinition of “Glucoamylase activity (AGU)”.

In a particular embodiment, the alpha-amylase is selected from thealpha-amylase disclosed in SEQ ID NO: 2 or an alpha-amylase having asequence identity to the polypeptide of SEQ ID NO: 2 or the maturepolypeptide thereof, of at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which has alpha-amylase activity; e.g., at least75%, such as at least 80%, at least 85%, at least 90% or such as atleast 95% of the alpha-amylase activity of the alpha-amylase disclosedin SEQ ID NO: 2 or the mature polypeptide thereof, when tested as setforth above in relation to the definition of “Acid alpha-Amylase Units(FAU(A))”. In another particular embodiment the alpha-amylase isselected from the alpha-amylases disclosed in SEQ ID NO: 5 or analpha-amylase having a sequence identity to the polypeptide of SEQ IDNO: 5 or the mature polypeptide thereof, of at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which has alpha-amylase activity;e.g., at least 75%, such as at least 80%, at least 85%, at least 90% orsuch as at least 95% of the alpha-amylase activity of the alpha-amylasedisclosed in SEQ ID NO: 5 or the mature polypeptide thereof, when testedas set forth above in relation to the definition of “Acid alpha-AmylaseUnits (FAU(A))”.

In a particular embodiment, the pullulanase is selected from thepullulanase disclosed in SEQ ID NO: 3 or the mature polypeptide thereof,and a pullulanase having a sequence identity to the polypeptide of SEQID NO: 3 or the mature polypeptide thereof, of at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%, which has pullulanaseactivity; e.g., at least 75%, such as at least 80%, at least 85%, atleast 90% or such as at least 95% of the pullulanase activity of theamino acid sequence set forth in SEQ ID NO: 3 or a mature polypeptidethereof, when tested as set forth above in relation to the definition of“Pullulanase activity (NPUN)”.

In a further specific embodiment, the composition comprises

i) a glucoamylase is selected from the glucoamylases disclosed in SEQ IDNO: 1 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 1 of at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which hasglucoamylase activity; e.g., at least 75%, such as at least 80%, atleast 85%, at least 90% or such as at least 95% of the glucoamylaseactivity of the glucoamylases disclosed in SEQ ID NO: 1 or the maturepolypeptide thereof, when tested as set forth above in relation to thedefinition of “Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 2 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 2 of at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which hasalpha-amylase activity; e.g., at least 75%, such as at least 80%, atleast 85%, at least 90% or such as at least 95% of the alpha-amylaseactivity of the alpha-amylase disclosed in SEQ ID NO: 2 or the maturepolypeptide thereof, when tested as set forth above in relation to thedefinition of “Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which has pullulanaseactivity; e.g., at least 75%, such as at least 80%, at least 85%, atleast 90% or such as at least 95% of the pullulanase activity of theamino acid sequence set forth in SEQ ID NO: 3 or a mature polypeptidethereof, when tested as set forth above in relation to the definition of“Pullulanase activity (NPUN)”.

Particularly, the composition may comprise:

i) a glucoamylase selected from the glucoamylase disclosed in SEQ ID NO:1 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 1 or themature polypeptide thereof, of at least 90% which has glucoamylaseactivity; e.g., at least 90% of the glucoamylase activity of theglucoamylase disclosed in SEQ ID NO: 1 or the mature polypeptidethereof, when tested as set forth above in relation to the definition of“Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 2 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 2 or themature polypeptide thereof of at least 90%, which has alpha-amylaseactivity; e.g., at least 90% of the alpha-amylase activity of thealpha-amylase disclosed in SEQ ID NO: 2 or the mature polypeptidethereof, when tested as set forth above in relation to the definition of“Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 or the maturepolypeptide thereof of at least 90%, which has pullulanase activity;e.g., at least 90% of the pullulanase activity of the amino acidsequence set forth in SEQ ID NO: 3 or a mature polypeptide thereof, whentested as set forth above in relation to the definition of “Pullulanaseactivity (NPUN)”.

Particularly, the composition may comprise:

i) a glucoamylase selected from the glucoamylase disclosed in SEQ ID NO:1 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the glucoamylase of SEQ ID NO: 1 or the maturepolypeptide thereof of at least 95% which has glucoamylase activity;e.g., at least 95% of the glucoamylase activity of the glucoamylasesdisclosed in SEQ ID NO: 1 or the mature polypeptide thereof, when testedas set forth above in relation to the definition of “Glucoamylaseactivity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 2 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 2 of at least95%, which has alpha-amylase activity; e.g., at least 95% of thealpha-amylase activity of the alpha-amylase disclosed in SEQ ID NO: 2 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 95%,which has pullulanase activity; e.g., at least 95% of the pullulanaseactivity of the amino acid sequence set forth in SEQ ID NO: 3 or themature polypeptide thereof, when tested as set forth above in relationto the definition of

“Pullulanase activity (NPUN)”.

Particularly, the composition may comprise:

i) a glucoamylase selected from the glucoamylase disclosed in SEQ ID NO:1 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 1 of at least97% which has glucoamylase activity; e.g., at least 95% of theglucoamylase activity of the glucoamylases disclosed in SEQ ID NO: 1 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 2 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 2 of at least97%, which has alpha-amylase activity; e.g., at least 95% of thealpha-amylase activity of the alpha-amylase disclosed in SEQ ID NO: 2 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 97%,which has pullulanase activity; e.g., at least 95% of the pullulanaseactivity of the amino acid sequence set forth in SEQ ID NO: 3 or amature polypeptide thereof, when tested as set forth above in relationto the definition of “Pullulanase activity (NPUN)”.

Particularly, the composition may comprise:

i) a glucoamylase selected from the glucoamylases disclosed in SEQ IDNO: 1 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 1 of at least99% which has glucoamylase activity; e.g., at least 95% of theglucoamylase activity of the glucoamylase disclosed in SEQ ID NO: 1 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 2 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 2 of at least99%, which has alpha-amylase activity; e.g., at least 95% of thealpha-amylase activity of the alpha-amylase disclosed in SEQ ID NO: 2 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 99%,which has pullulanase activity; e.g., at least 95% of the pullulanaseactivity of the amino acid sequence set forth in SEQ ID NO: 3 or amature polypeptide thereof, when tested as set forth above in relationto the definition of “Pullulanase activity (NPUN)”.

In a further specific embodiment, the composition comprises:

i) a glucoamylase is selected from the glucoamylase disclosed in SEQ IDNO: 4 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 4 of at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which hasglucoamylase activity; e.g., at least 75%, such as at least 80%, atleast 85%, at least 90% or such as at least 95% of the glucoamylaseactivity of the glucoamylase disclosed in SEQ ID NO: 4 or the maturepolypeptide thereof, when tested as set forth above in relation to thedefinition of “Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 5 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 5 of at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100%, which hasalpha-amylase activity; e.g., at least 75%, such as at least 80%, atleast 85%, at least 90% or such as at least 95% of the alpha-amylaseactivity of the alpha-amylase disclosed in SEQ ID NO: 5 or the maturepolypeptide thereof, when tested as set forth above in relation to thedefinition of “Acid alpha-Amylase Units (FAU(A))”, and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which has pullulanaseactivity; e.g., at least 75%, such as at least 80%, at least 85%, atleast 90% or such as at least 95% of the pullulanase activity of theamino acid sequence set forth in SEQ ID NO: 3 or a mature polypeptidethereof, when tested as set forth above in relation to the definition of“Pullulanase activity (NPUN)”.

Particularly, the composition may comprise:

i) a glucoamylase selected from the glucoamylase disclosed in SEQ ID NO:4 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 4 of at least90% which has glucoamylase activity, e.g., at least 90% of theglucoamylase activity of the glucoamylase disclosed in SEQ ID NO: 4 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 5 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 5 of at least90%, which has alpha-amylase activity; e.g., at least 95% of thealpha-amylase activity of the alpha-amylase disclosed in SEQ ID NO: 5 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 90%,which has pullulanase activity, e.g., at least 90% of the pullulanaseactivity of the amino acid sequence set forth in SEQ ID NO: 3 or amature polypeptide thereof, when tested as set forth above in relationto the definition of “Pullulanase activity (NPUN)”.

Particularly, the composition may comprise:

i) a glucoamylase selected from the glucoamylase disclosed in SEQ ID NO:4 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 4 of at least95% which has glucoamylase activity, e.g., at least 95% of theglucoamylase activity of the glucoamylase disclosed in SEQ ID NO: 4 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 5 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 5 of at least95%, which has alpha-amylase activity; e.g., at least 95% of thealpha-amylase activity of the alpha-amylase disclosed in SEQ ID NO: 5 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 95%,which has pullulanase activity; e.g., at least 95% of the pullulanaseactivity of the amino acid sequence set forth in SEQ ID NO: 3 or amature polypeptide thereof, when tested as set forth above in relationto the definition of “Pullulanase activity (NPUN)”.

Particularly, the composition may comprise:

i) a glucoamylase selected from the glucoamylase disclosed in SEQ ID NO:4 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 4 of at least97% which has glucoamylase activity, e.g., at least 95% of theglucoamylase activity of the glucoamylase disclosed in SEQ ID NO: 4 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 5 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 5 of at least97%, which has alpha-amylase activity; e.g., at least 95% of thealpha-amylase activity of the alpha-amylase disclosed in SEQ ID NO: 5 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 97%,which has pullulanase activity, e.g., at least 95% of the pullulanaseactivity of the amino acid sequence set forth in SEQ ID NO: 3 or amature polypeptide thereof, when tested as set forth above in relationto the definition of “Pullulanase activity (NPUN)”.

Particularly, the composition may comprise:

i) a glucoamylase selected from the glucoamylases disclosed in SEQ IDNO: 4 or the mature polypeptide thereof, and a glucoamylase having asequence identity to the mature polypeptide of SEQ ID NO: 4 of at least99% which has glucoamylase activity, e.g., at least 95% of theglucoamylase activity of the glucoamylase disclosed in SEQ ID NO: 4 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Glucoamylase activity (AGU)”;

ii) an alpha-amylase selected from the alpha-amylase disclosed in SEQ IDNO: 5 or the mature polypeptide thereof, and an alpha-amylase having asequence identity to the mature polypeptide of SEQ ID NO: 5 of at least99%, which has alpha-amylase activity; e.g., at least 95% of thealpha-amylase activity of the alpha-amylase disclosed in SEQ ID NO: 5 orthe mature polypeptide thereof, when tested as set forth above inrelation to the definition of “Acid alpha-Amylase Units (FAU(A))”; and

iii) a pullulanase selected from a pullulanase disclosed in SEQ ID NO: 3or the mature polypeptide thereof, and a pullulanase having a sequenceidentity to the mature polypeptide of SEQ ID NO: 3 of at least 99%,which has pullulanase activity; e.g., at least 95% of the pullulanaseactivity of the amino acid sequence set forth in SEQ ID NO: 3 or amature polypeptide thereof, when tested as set forth above in relationto the definition of “Pullulanase activity (NPUN)”.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Uses

The composition according to the invention may be used in asaccharification process to produce glucose syrup. Therefore, in afurther aspect, the invention relates to a method of making glucosesyrup from liquefied starch comprising, contacting the liquefied starchwith a composition according to the invention.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Material and Methods

Glucoamylase Activity

Glucoamylase Activity (AGU)

The Glucoamylase Unit (AGU) is defined as the amount of enzyme, whichhydrolyzes 1 micromole maltose per minute under the standard conditions(37° C., pH 4.3, substrate: maltose 100 mM, buffer: acetate 0.1 M,reaction time 6 minutes as set out in the glucoamylase incubationbelow), thereby generating glucose.

glucoamylase incubation: Substrate: maltose 100 mM Buffer: acetate 0.1MpH: 4.30 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 6minutes Enzyme working range: 0.5-4.0 AGU/mL

The analysis principle is described by 3 reaction steps:

Step 1 is an enzyme reaction:

Glucoamylase (AMG), EC 3.2.1.3 (exo-alpha-1,4-glucan-glucohydrolase),hydrolyzes maltose to form alpha-D-glucose. After incubation, thereaction is stopped with NaOH.

Steps 2 and 3 result in an endpoint reaction:

Glucose is phosphorylated by ATP, in a reaction catalyzed by hexokinase.The glucose-6-phosphate formed is oxidized to 6-phosphogluconate byglucose-6-phosphate dehydrogenase. In this same reaction, an equimolaramount of NAD+ is reduced to NADH with a resulting increase inabsorbance at 340 nm. An autoanalyzer system such as Konelab 30 Analyzer(Thermo Fisher Scientific) may be used.

Color reaction Tris approx. 35 mM ATP 0.7 mM NAD⁺ 0.7 mM Mg²⁺ 1.8 mMHexokinase >850 U/L Glucose-6-P-DH >850 U/L pH approx. 7.8 Temperature37.0° C. ± 1.0° C. Reaction time 420 seconds Wavelength 340 nmAcid Alpha-Amylase Activity

When used according to the present invention, the activity of any acidalpha-amylase may be measured in FAU(A) (Acid Fungal Alpha-amylaseUnits).

Acid Alpha-Amylase Activity (FAU(A))

Acid alpha-amylase activity may be measured in FAU(A) (Acid FungalAlpha-amylase Units). 1 FAU(A) is defined as the amount of enzyme whichdegrades 5.260 mg starch dry matter per hour under standard conditions.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

FAU(A), the acid alpha-amylase activity is determined in accordance withthe following description, and is measured relative to a Novozymesstandard which is available on request from Novozymes A/S, Denmark. Theprinciple of the reaction is based on the two steps. In the first stepthe enzyme acid alpha-amylase hydrolyzes starch into differentoligosaccharides. In the second step iodine forms a blue complex withstarch but not with its degradation products. The intensity of color istherefore directly proportional to the concentration of starch. Theactivity is determined using reverse colorimetry as a reduction in theconcentration of starch under specified analytic conditions.

First reaction, starch degradation Substrate Starch, approx. 0.3 g/LBuffer Citrate, approx. 0.05M CaCl2, 1.85 mM pH 2.50 ± 0.05 Incubationtemperature 37° C. Reaction time 180 seconds Enzyme working range0.01-0.04 FAU(A)/mL

Second reaction, starch-iodine complex Iodine 0.0432 g/L Incubationtemperature 37° C. Reaction time 60 seconds Wavelength 600 nm

If further details are preferred, these can be found in EB-SM-0510.02available on request from Novozymes A/S, Denmark, and incorporated byreference

Pullulanase Activity

Endo-pullulanases hydrolyze α-1,6-glycosidic bonds in pullulan (−BH4reduced to reduce background reducing sugar), releasing maltotrioseunits with reducing carbohydrate ends. Pullulanase is a pullulan6-glucano-hydrolase with the enzyme classification number E.C.3.2.1.41.

The NPUN (New Pullulanase Unit Novozymes) is a unit of endopullulanaseactivity measured in the following procedure, and is measured relativeto a Novozymes standard which is available on request from NovozymesA/S, Denmark.

1 NPUN=One pullulanase unit (NPUN) is defined as the enzyme amount,which releases reducing ends equivalent to 0.35 μmol glucose per minuteunder the standard conditions.

In the first reaction, the substrate is equally present in both samplemain and sample blank. However, the reaction of sample main is performedat pH 5.0, while there is no reaction in the sample blank at pH 9.6,where neither pullulanases nor amyloglucosidases (glucoamylase) areenzymatically active.

First reaction, pullulan degradation Substrate BH4 reduced pullulan, 5.3g/L Buffer (main) Acetate, approx. 0.1M EDTA, 5.3 mM Acarbose, 0.018%(if sample contains glucoamylase) pH (main) 5.0 Buffer (blank) CHES, 42mM acetate, 17 mM EDTA, 5.3 mM pH (blank) 9.6 Incubation temperature 50°C. Reaction time 540 seconds Enzyme working range 0.03-0.15 NPUN/mL

In the second reaction, the pH was adjusted to approx. 9.6 and theglucose in samples is phosphorylated to non-reducingD-glucose-6-phosphate by glucokinase, which has optimal activity andstability in this range and is specific to glucose at pH 9 (ref. Goward,1986, Biochem. J. 237: 415-420). This step depends on identical pH insample main and sample blank to remove equal amounts of glucose in both.

Second reaction, background glucose elimination Substrate glucose insample, after first reaction Buffer CHES, 58 mM (main) or 76 mM (blank)acetate, 43 mM (main) or 7.2 mM (blank) EDTA, 2.2 mM ATP, 1.11 mg/mlMgCl₂, 4.4 mM Glucokinase 0.11 U/ml pH approx. 9.6 Incubationtemperature 50° C. Reaction time 720 seconds

The second reaction is stopped by and alkaline reagent >pH 11 containingPAHBAH (p-Hydroxy benzoic acid hydrazide) and bismuth, which complexeswith reducing sugars to produce color detected at 405 nm. The producedcolor is proportional to the pullulanase activity.

Third reaction, PAHBAH-Bi reaction Substrate maltotriose formed bypullulanase, after second reaction PAHBAH 56 mM Tartrate 75 mM Bi³ 6.0mM NaOH 195 mM pH alkaline Incubation temperature 50° C. Reaction time1000 seconds Wavelength 405 nm

If further details are preferred, these can be found in 2010-28835-02available on request from Novozymes A/S, Denmark, and incorporated byreference.

Example 1

Maltodextrin which dextrose equivalent (DE) was adjusted to 11 wasprepared from a conventional starch liquefaction process using cornstarch and spray-dried for this experiment. The maltodextrin powder wasdissolved in milliQ water and the pH was adjusted by HCl/NaOH to be 4.3at 60° C., and then the solid was adjusted to 33% dry solid (DS) bymeasuring refractive index (RI) of the syrup. Saccharification wasstarted by mixing 18 g maltodextrin solution and 2 ml enzyme mixturecontaining Aspergillus niger glucoamylase (SEQ ID NO: 1), pullulanase(Promozyme D2®) (SEQ ID NO: 3), and Aspergillus niger acid alpha-amylase(SEQ ID NO: 2) at different dosages. The samples were incubated at 60°C. with stirring and were taken at different time intervals fordetermination of sugar component. The enzyme dosages and the DP1-DP4+compositions at the time point that DP1 fraction is maximized were shownin Table 1.

A. niger alpha- A. niger amylase glucoamylase (SEQ ID Promozyme (SEQ IDNO: 2) D2 ® (SEQ Peak NO: 1) FAU(A)/ ID NO: 3) NPUN/ time DP1 DP2 DP3DP4+ AGU/gDS gDS NPUN/gDS FAU(A) hr % % % % Comment 0.18 0.045 0.34 7.5648 95.6 2.4 0.5 1.4 Dextrozyme 0.18 0.011 0.34 30.22 72 95.3 2.8 0.4 1.5DX ® 0.18 0.023 0.34 15.11 60 95.4 2.6 0.5 1.5 0.18 0.045 0.34 7.56 4895.5 2.4 0.6 1.5 0.18 0.09 0.34 3.78 48 95.4 2.4 0.7 1.5 0.18 0.18 0.341.89 48 95.4 2.4 0.7 1.5 0.18 0.011 1.01 89.78 36 95.9 2.1 0.6 1.4 0.180.023 1.01 44.89 48 95.8 2.4 0.6 1.3 0.18 0.045 1.01 22.44 36 95.6 2.20.8 1.5 0.18 0.09 1.01 11.22 60 95.5 2.6 0.6 1.3 0.18 0.18 1.01 5.61 4895.5 2.4 0.8 1.4

Table 1 shows that the enzyme blends with NPUN/FAU(A) ratio higher than44.89 showed higher DP1 fraction than Dextrozyme DX®. Even at 3-timeshigher NPUN activity (1.01 NPUN/gDS) than Dextrozyme DX, the peak DP1was not as high as Dextrozyme DX at higher FAU(A) activity than 0.045.

Example 2

Saccharification Using Different Enzyme Blends

Maltodextrin powder from corn starch liquefaction was dissolved in waterwhile heating to make slurry at 34.4% dry solids. The solid content ofthe slurry was measured using Refractive index measurement showing1.39271. The slurry was adjusted to a pH of 4.3 using a 1 M Hydrochloricacid solution. 18 gram aliquots of this slurry were added to 18 glassreaction scintillation vials with septum cap closures and were insertedin a heating block to be heated to a temperature of 61° C. Each vial wasgiven an enzyme dosage based on the table below and additional water wasadded to each vial to reach a target dry solid of 33%. The enzyme blendcomprised a glucoamylase derived from Gloeophyllum trabeum (SEQ ID NO:4), an alpha-amylase derived from Rhizomucor pusillus (SEQ ID NO: 5) anda pullulanase derived from Bacillus deramificans (SEQ ID NO: 3). 1.5 mLsamples were taken via needles through the septum from each vial atdifferent time points (36 hours, 42 hours, 48 hours, 54 hours and 60hours) and were deactivated at 105° C. for 5 minutes. 1 mL of eachdeactivated sample was diluted with 4 mL deionized water. The dilutedsamples were evaluated using a HPLC method DP1-4 for measuring dextrosepurity (% DP1 or % DX).

Results from Table 2 show the higher the NPUN/FAU(A) ratio, the higherthe percent dextrose.

TABLE 2 GA PUL SEQ ID AA SEQ SEQ ID NO: 4 ID NO: 5 NO: 3 Dose Dose Dose(AGU/g (FAU(A)/ (NPUN/ NPUN/FAU(A) % DX % DX % DX % DX % DX DS) g DS) gDS) ratio 36 hr 42 hr 48 hr 54 hr 60 hr 0.18 0 0.72 N/A 92.2 93.5 94.695.3 95.8 0.18 0 1.08 N/A 93.5 94.8 95.6 96.1 96.5 0.18 0.008 0.9 112.594.1 95.2 95.5 95.8 95.8 0.18 0.008 0.9 112.5 94.4 95.3 95.7 95.9 96.00.18 0.015 0.72 48 93.6 94.8 95.3 95.5 95.6 0.18 0.015 1.08 72 94.0 94.995.3 95.5 95.6 0.23 0 0.9 N/A 94.1 95.0 95.6 96.0 96.3 0.23 0.008 0.7290 95.5 95.8 96.0 96.0 96.0 0.23 0.008 0.9 112.5 95.5 95.8 95.9 96.096.0 0.23 0.008 1.08 135 95.7 96.0 96.0 96.0 96.1 0.23 0.008 1.08 13595.7 95.9 96.0 96.1 96.0 0.23 0.015 0.9 60 95.3 95.7 95.8 95.8 95.9 0.230.015 0.9 60 95.0 95.5 95.6 95.8 95.7 0.28 0 0.72 N/A 94.6 95.5 95.896.1 96.1 0.28 0 1.08 N/A 95.7 96.3 96.3 96.6 96.4 0.28 0.008 0.9 112.595.9 96.0 96.0 96.0 96.0 0.28 0.015 0.72 48 95.8 95.9 95.9 95.9 95.90.28 0.015 1.08 72 95.7 95.7 95.8 95.9 95.8

In a different experiment, performed according to the same procedure asabove, results are shown in Table 3.

TABLE 3 AA SEQ ID GA SEQ ID NO: 5 AA PUL SEQ ID NO: 4 % DX % DX % DXdose NO: 3 dose AMG dose NPUN/AFAU at % DX at at at (AFAU/gDS)(NPUN/gDS) (AGU/gDS) Ratio 36 hr 42 hr 48 hr 60 hr 0.0 0.96 0.25 N/A96.2 96.6 96.7 96.8 0.001 0.96 0.25 960 96.8 96.7 96.7 96.6 0.002 0.960.25 480 96.6 96.6 96.6 96.5 0.003 0.96 0.25 320 96.3 96.4 96.5 96.30.005 0.96 0.25 191 96.2 96.3 96.2 96.2

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. A composition comprising an alpha-amylase,a pullulanase and a glucoamylase, wherein the alpha-amylase has at least85% sequence identity to SEQ ID NO: 2 or a mature polypeptide thereof,the pullulanase has at least 85% sequence identity to SEQ ID NO: 3 or amature polypeptide thereof, and the glucoamylase has at least 85%sequence identity to SEQ ID NO: 1 or a mature polypeptide thereof; andwherein the ratio of pullulanase dose expressed as New Pullulanase UnitsNovozymes (NPUN)/gDS, to alpha-amylase dose expressed as Acid FungalAlpha-amylase Units (FAU(A))/g or as KNU/g is at least 60 and the ratioof pullulanase dose in NPUN/gDS to glucoamylase dose in GlucoamylaseUnits (AGU)/gDS is in the range of 2-15.
 2. The composition of claim 1,wherein the alpha-amylase has at least 90% sequence identity to SEQ IDNO: 2 or a mature polypeptide thereof, the pullulanase has at least 90%sequence identity to SEQ ID NO: 3 or a mature polypeptide thereof, andthe glucoamylase has at least 90% sequence identity to SEQ ID NO: 1 or amature polypeptide thereof.
 3. The composition of claim 1, wherein thealpha-amylase has at least 95% sequence identity to SEQ ID NO: 2 or amature polypeptide thereof, the pullulanase has at least 95% sequenceidentity to SEQ ID NO: 3 or a mature polypeptide thereof, and theglucoamylase has at least 95% sequence identity to SEQ ID NO: 1 or amature polypeptide thereof.
 4. The composition of claim 1, wherein thealpha-amylase has the amino acid sequence of SEQ ID NO: 2 or a maturepolypeptide thereof, the pullulanase has the amino acid sequence of SEQID NO: 3 or a mature polypeptide thereof, and the glucoamylase has theamino acid sequence of SEQ ID NO: 1 or a mature polypeptide thereof. 5.The composition of claim 1, wherein the ratio of pullulanase doseexpressed as NPUN/g, to alpha-amylase dose expressed as FAU(A)/g or asKNU/g is at least
 75. 6. The composition of claim 1, wherein the ratioof pullulanase dose expressed as NPUN/g, to alpha-amylase dose expressedas FAU(A)/g or as KNU/g is in the range from 60-1000.
 7. The compositionof claim 6, wherein the ratio between pullulanase expressed as NPUN/gand glucoamylase expressed as AGU/g is at least
 2. 8. A method of makingglucose syrup, comprising saccharifying the liquefied starch with acomposition of claim
 1. 9. A method of making glucose syrup, comprisingsaccharifying the liquefied starch with a composition of claim
 2. 10. Amethod of making glucose syrup, comprising saccharifying the liquefiedstarch with a composition of claim
 3. 11. A method of making glucosesyrup, comprising saccharifying the liquefied starch with a compositionof claim
 4. 12. The method of claim 8, wherein the glucoamylase doseexpressed as AGU/gDS is at least 0.1.
 13. The method of claim 8, whereinthe initial dry solids content (DS) in the liquefied starch substrate isat least 25%.
 14. The method of claim 8, wherein saccharification timeis at least 24 hours.
 15. The composition of claim 1, wherein thealpha-amylase has at least 96% sequence identity to SEQ ID NO: 2 or amature polypeptide thereof, the pullulanase has at least 96% sequenceidentity to SEQ ID NO: 3 or a mature polypeptide thereof, and theglucoamylase has at least 96% sequence identity to SEQ ID NO: 1 or amature polypeptide thereof.
 16. The composition of claim 1, wherein thealpha-amylase has at least 97% sequence identity to SEQ ID NO: 2 or amature polypeptide thereof, the pullulanase has at least 97% sequenceidentity to SEQ ID NO: 3 or a mature polypeptide thereof, and theglucoamylase has at least 97% sequence identity to SEQ ID NO: 1 or amature polypeptide thereof.
 17. The composition of claim 1, wherein thealpha-amylase has at least 98% sequence identity to SEQ ID NO: 2 or amature polypeptide thereof, the pullulanase has at least 98% sequenceidentity to SEQ ID NO: 3 or a mature polypeptide thereof, and theglucoamylase has at least 98% sequence identity to SEQ ID NO: 1 or amature polypeptide thereof.
 18. The composition of claim 1, wherein thealpha-amylase has at least 99% sequence identity to SEQ ID NO: 2 or amature polypeptide thereof, the pullulanase has at least 99% sequenceidentity to SEQ ID NO: 3 or a mature polypeptide thereof, and theglucoamylase has at least 99% sequence identity to SEQ ID NO: 1 or amature polypeptide thereof.